Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica.
Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione. The pi-class GST cDNA (leGSTp) was cloned from the cold-adapted Antarctic bivalve Laternula elliptica. We used degenerated primers designed based on highly conserved regions of known mollusk GSTs to amplify the corresponding L. elliptica mRNA. Full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full sequence of the GST cDNA was 1189 bp in length, with a 5' untranslated region (UTR) of 74 bp, a 3' UTR of 485 bp, and an open reading frame of 630 bp encoding 209 amino acid residues with an estimated molecular mass of 23.9 kDa and an estimated isoelectric point of 8.3. Quantitative RT-PCR confirmed basal expression of leGSTp, which was up-regulated upon heat treatment (10 degrees C for different time periods) by a factor of 2.3 (at 24 h) and 2.7 (at 48 h) in the digestive gland and gill tissues, respectively. The recombinant leGSTp expressed in Escherichia coli was purified by affinity chromatography and characterized. The purified leGSTp exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). The recombinant leGSTp had a maximum activity at approximately pH 8.0, and its optimum temperature was 35 degrees C.
Kim
Meesun
M
Korea Polar Research Institute, Korea Ocean Research and Development Institute, Songdo-dong 7-50, Yeonsu-gu, Incheon 406-840, South Korea.
Ahn
In-Young
IY
Cheon
Jina
J
Park
Hyun
H
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000818:Animals; D000864:Antarctic Regions; D001483:Base Sequence; D049872:Bivalvia / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003080:Cold Temperature; D004064:Digestive System / Q000201:enzymology; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004790:Enzyme Induction; D004976:Ethacrynic Acid / Q000378:metabolism; D005880:Gills / Q000201:enzymology; D051549:Glutathione S-Transferase pi / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013312:Stress, Physiological; D013379:Substrate Specificity
D004137:Dinitrochlorobenzene; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051549:Glutathione S-Transferase pi; D004976:Ethacrynic Acid